FLASH effect on the liver and spleen of mice—a transcriptional perspective

Background FLASH radiotherapy (FLASH‐RT), delivered at an ultra‐high dose rate (typically ≥ 40 Gy/s), has been shown to reduce normal tissue toxicity compared with conventional dose‐rate radiotherapy (CONV‐RT). However, the transcriptomic mechanisms underlying FLASH‐mediated protection remain incompletely understood. Purpose This study aimed to compare the biological effects of FLASH and CONV irradiation on mouse liver and spleen tissues and to identify molecular pathways associated with the FLASH effect from a transcriptional perspective. Methods C57BL/6J male mice received a single 12 Gy abdominal dose delivered at either 2 Gy/min (CONV) or 100 Gy/s (FLASH). Twelve days after irradiation, spleen and liver tissues were harvested for histological section analysis and RNA‐seq. Inflammatory responses in the spleen and liver were assessed by hematoxylin‐eosin staining (H&E staining) and immunohistochemical quantification. A series of bioinformatic analyses, such as gene enrichment, protein–protein interaction (PPI) analysis, and transcription factor prediction, were performed to explore the effect of FLASH irradiation. Subsequently, reverse transcription quantitative PCR (RT‐qPCR), western blot analysis, and immunohistochemical quantification were used to verify the bioinformatic results. Results Histological and immunohistochemical analyses showed less prominent inflammatory changes and lower immune‐cell marker staining in mouse spleen and liver after FLASH than after CONV irradiation at 12 days post‐irradiation. Bioinformatic analysis and corresponding experimental results revealed that compared to CONV irradiation, FLASH irradiation induced down‐regulation of oxidative phosphorylation and cytoplasmic translation pathways in both mouse liver and spleen. Meanwhile, immune‐related pathways, especially interferon β (IFNβ) signaling pathways, were significantly activated in both tissues after FLASH than after CONV irradiation. For these three pathways, key transcription factors were further identified and verified. Notably, STAT1 was found to play a potentially critical role in the upregulation of the IFNβ‐related pathway under FLASH irradiation conditions in both liver and spleen. Conclusions FLASH irradiation induces distinct metabolic and immune adaptations in normal mouse liver and spleen tissues compared with CONV irradiation. The coordinated suppression of metabolic and translational pathways, together with activation of IFNβ‐STAT1 signaling program, provides transcriptomic evidence for potential mechanisms underlying FLASH‐mediated normal tissue sparing. These findings offer candidate pathways and molecular markers for future functional validation and support the clinical translation of FLASH‐RT.